P469: ACUTE MYELOID LEUKEMIA DRIVES BCL-2 UPREGULATION IN NON MALIGNANT HSPCS WHICH IS TARGETED BY VENETOCLAX AND CAUSES CYTOPENIA
Authors: Dominic Fowler-Shorten, Rebecca Maynard, Katherine Hampton, Martha Ehikioya, Ravindu De Silva, Kristian Bowles, Stuart Rushworth, Charlotte Hellmich
Journal: HemaSphere (2023)

Background: The BH3 mimetic Venetoclax, in combination with low dose cytarabine, decitabine or azacitidine, has shown clinical efficacy in newly diagnosed acute myeloid leukemia (AML) patients over 75 or those ineligible for intensive induction chemotherapy. This has been a significant advance, particularly in the treatment of older AML patients who historically have been difficult to treat. Venetoclax selectively inhibits the BCL-2 protein which is overexpressed in AML in order to reactivate intrinsic apoptosis. However, this treatment regime is associated with cytopenias including neutropenia, febrile neutropenia, and thrombocytopenia which leaves patients immunocompromised. The underlying cause of cytopenia in the context of Venetoclax-treated AML remains unknown Aims: Here, we hypothesise that Venetoclax depletes haematopoietic stem and progenitor cells (HSPCs) in AML leading to cytopenia. Using in vitro and in vivo modelling, we aim to investigate the mechanism of cytopenia in Venetoclax-treated AML. Methods: Two established AML models, HOXA9/Meis1 and MN1, were used to assess the BCL-2 expression profile in HSPCs. Isolated Lineage negative Sca positive and cKit positive (LSK) cells were co-cultured in transwell inserts with either HOXA9/Meis1 or MN1 cells and qPCR assessed for BCL-2 expression. The MN1 cells were injected into C57Bl/6 mice followed by Venetoclax treatment by oral gavage. Bloods were assessed by flow cytometry before and after treatment, and bone marrow was extracted following sacrifice for flow cytometry. HSPCs were FACS purified, and qPCR assessed for BCL-2 expression post-treatment. Mechanisms of BCL-2 expression were explored using the co-culture system and targeted inhibitors. Results: We identified BCL-2 overexpression in LSKs co-cultured with either HOXA9/Meis1 and MN1 compared to HSPC only controls. In vivo analysis of BCL-2 in LSKs from AML engrafted mice showed increased BCL-2 expression confirming the in vitro data. Next, we confirmed that Venetoclax caused cytopenia in AML engrafted mice. We also show that Venetoclax-treated AML mice had reduced numbers of HSPCs compared to untreated AML mice. Finally, we wanted to understand the mechanism of AML-induced BCL-2 expression in HSPCs. We found that interleukin-3 (IL-3) activates the BCL-2 transcription in HSPCs. Summary/Conclusion: Here, we demonstrate that BCL-2 is overexpressed in HSPCs during AML progression both in vitro and in vivo using two AML models. These data also show that Venetoclax depletes HSPCs in AML models suggesting that this is the cause of the cytopenias. Finally, we show that elevated IL-3 levels associated with AML cause the increased BCL-2 expression in HSPCs. This finding could lead to a new combination therapy for AML involving Venetoclax that reduces the incidence of cytopenia currently experienced by patients. Keywords: BCL2, AML, Neutropenia, Venetoclax